Comparison of CX-4945 and SGC-CK2-1 as inhibitors of CSNK2 using quantitative phosphoproteomics: Triple SILAC in combination with inhibitor-resistant CSNK2

Abstract

Specificity is a limiting factor when using small-molecule inhibitors to study protein kinase signalling. Since inhibitor-resistant kinase mutants (i.e., drug-resistant alleles) remain active in the presence of inhibitor, they facilitate validation of on-target effects. By combining an inhibitor-resistant kinase mutant with mass spectrometry-based phosphoproteomics, we previously devised a systematic strategy for reliable identification and validation of CSNK2 substrates. In this study, we use the same strategy to evaluate the selectivity of CX-4945, a clinical stage CSNK2 inhibitor, and SGC-CK2-1, a chemical probe selectively targeting CSNK2. Human osteosarcoma (U2OS) cells expressing exogenous wild-type CSNK2A1 (WT) or an inhibitor-resistant triple mutant (TM, V66A/H160D/I174A) were treated with CX-4945 or SGC-CK2-1 prior to analysis using triple SILAC (phospho)proteomics. The minority of phosphosites, 15% at 4 ​h and 5% at 24 ​h, that were significantly downregulated in response to CX-4945 treatment were determined to be CSNK2A1-dependent. By comparison, the majority of phosphosites, >55% at both 4 and 24 ​h, that were significantly downregulated in response to SGC-CK2-1 were identified as CSNK2A1-dependent. This indicates that SGC-CK2-1 exhibits significantly greater selectivity towards CSNK2A1 than CX-4945. Notably, utilization of SGC-CK2-1 in cells expressing CSNK2A1-TM enabled the identification of >300 CSNK2A1-dependent phosphosites. Overall, this study highlights the utility of exploiting highly selective chemical probes together with inhibitor-resistant kinase mutants to facilitate identification of bona fide kinase substrates.